Human tissue inhibitor of metalloproteinase-4

ABSTRACT

A human tissue inhibitor of metalloproteinases-4 polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques. Also disclosed are methods for utilizing such polypeptide for the treatment of diseases, including arthritis and cancer. Antagonists against such polypeptides and their use as a therapeutic to resorb scar tissue are also disclosed. Diagnostic assays for detecting levels of human TIMP-4 protein and mutations in human TIMP-4 nucleic acid sequence are also disclosed.

This application is a Divisional of and claims priority under 35 U.S.C.section 120 to patent application Ser. No. 08/463,261, filed Jun. 5,1995, pending, which is a continuation U.S. patent application Ser. No.PCT US94/14498 filed Dec. 13, 1994, both of which are incorporatedherein by reference in their entireties.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptides of the presentinvention are human tissue inhibitor of metalloproteinase-4polypeptides, hereinafter referred to as “human TIMP-4”. The inventionalso relates to inhibiting the action of such polypeptides.

The extracellular matrix is a complex structure that contains collagen,proteoglycan, glycosaminoglycan, glycoproteins (fibronectin,chondronectin, laminin) and in some tissues, elastin (Hay, E. D., J.Cell Biol., 91:205-223 (1981)).

Matrix metalloproteinases (MMP's) constitute the major group ofzinc-binding endopeptidases that degrade extracellular matrix proteins,for example connective tissue, collagen and gelatin, during remodelingof connective tissue during normal physiological and some pathologicalprocesses. The unrestrained activity of MMP's may result in extensivetissue damage, and these enzymes have been implicated in a variety ofdisease processes, including tumor cell invasion, tumor angiogenesis andrheumatoid arthritis (Okada, Y., et al., J. Biol. Chem., 261:14245-14255(1986)). The MMP's are secreted from cells as inactive zymogens andtheir activity in the extracellular environment is regulated by variousactivators and inhibitors (Matrisian, L. M., Trends Genet., 6:121-125(1990)).

Regulation of metalloproteinase-mediated proteolysis may occur bynaturally occurring inhibitor proteins, such as tissue inhibitor ofmetalloproteinase (TIMP). The balance between the production andactivation of the MMP's, and their inhibition by natural inhibitors suchas TIMP, determines, in both physiological and pathological conditions,whether connective tissue is degraded.

MMP's include a number of proteases, exemplified by interstitial (typeI) collagenase itself, the stromelysins (also known as proteoglycanasesor transins), fibroblast and polymorphonuclear leukocyte gelatinases(also known as collagen-IV-ases), and ‘pump-1’ (putativemetalloproteases 1, uterine metalloproteases) [Goldberg et al, J. Biol.Chem. 2610:6600 (1986); Whitham et al, Biochem. J. 240:913 (1986);Breathnach et al, Nucleic Acids Res., 15:1139 (1987); Muller et al,Biochem. J., 253:187 (1988); Collier et al, J. Biol. Chem., 263:6579(1988); Murphy et al, Biochem. J., 258:463 (1989); Quantin et al,Biochem. (N.Y.), 28:5327 (1989); Birkedal-Hansen, J. Oral Pathol.,17:445 (1988)].

In general, the mammalian family of proteases has one or more of thefollowing properties: (a) optimal proteolytic activity around neutralpH; (b) dependence of the enzyme's activity on the presence of zinc, asevident by the loss of activity on treatment with divalent metal ionchelators, such as 1.10 phenanthroline (preferential chelation of zinc),or EDTA (less restricted chelating properties; EDTA and EGTA alsocontribute to enzyme inactivation via chelation of calcium ions requiredfor enzyme stability); (c) inhibition by TIMPs; (d) absence ofsignificant inhibition by known inhibitors of other families of neutral,zinc-containing metalloproteases, such as thermolysis,angiotensin-converting enzyme and ‘enkephalinases’; and (e) biosynthesisand secretion as latent precursor forms (zymogens), requiringextracellular activation. Activation has been achieved by a number ofendoproteases, organomercurials and chaotropic agents.

In general, members of the family of neutral metalloprotease enzymeshave distinctive substrate specificities. Thus, collagenase type I isunique in its ability to cleave a specific peptide bond within thenatural fibrils of the interstitial collagens (e.g. types I, II andIII). The gelatinases are only poorly active on these collagens, but areable to degrade denatured interstitial collagens, as well as thenon-fibrillar collagens, e.g. type IV, such as are found in the basementmembrane. Pump 1 has been reported to act preferentially on denaturedcollagens (gelatins), though its profile differs from that of thestromelysins or the collagenases type IV. Both the stromelysins and thegelatinases are also capable of degrading non-collagenous structuralproteins, such as the core protein of proteoglycan and elastin.Macromolecules involved in cell-to-substratum and cell-to-cellinteractions, such as laminin and fibronectin, are also susceptible todegradation by several of these metalloproteases.

Enzymes of this family are produced by synovial and skin fibroblasts,chondrocytes, peripheral mononuclear cells, keratinocytes and gingivaltissue, as well as existing within granule storage vesicles inpolymorphonuclear leukocytes (PMNLs).

Current information suggests that there is a family of metalloproteinaseinhibitors which comprises TIMP-1 (tissue inhibitor ofmetalloproteinases-1); TIMP-2; human TIMP-3 which has been cloned,expressed and mapped to human chromosome 22; and chicken tissueinhibitor of metalloproteinase (ChIMP-5). The polypeptide of the presentinvention has been putatively identified as a novel human TIMPpolypeptide based on amino acid sequence homology.

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is human TIMP-4, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding human TIMP-4,including mRNA's, DNA's, cDNA's, genomic DNA as well as biologicallyactive and diagnostically or therapeutically useful fragments, analogsand derivatives thereof.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques which comprises culturing recombinant prokaryotic and/oreukaryotic host cells, containing a human TIMP-4 nucleic acid sequenceunder conditions promoting expression of protein and subsequent recoveryof said protein.

In accordance with yet a further aspect of the present invention, thereis provided a method for treating conditions which are related toinsufficient human TIMP-4 activity which comprises administering to apatient in need thereof a pharmaceutical composition containing thehuman TIMP-4 protein of the invention which is effective to supplement apatient's endogenous human TIMP-4 and thereby alleviate said conditionswhich include, for example, arthritic diseases such as rheumatoid andosteoarthritis, soft tissue rheumatism, polychondritis and tendonitis;bone resorption diseases, such as osteoporosis, Paget's disease,hyperparathyroidism and cholesteatoma; the enhanced collagen destructionthat occurs in association with diabetes; the recessive classes ofdystrophic epidermolysis bullosa; periodontal disease, alveolitis andrelated consequences of gingival production of collagenase; cornealulceration; ulceration of the skin and gastro-intestinal tract andabnormal wound healing; post-operative conditions in which collagenaselevels are raised; cancer by blocking the destruction of tissue basementmembranes leading to cancer metastasis; demyelinating diseases of thecentral and peripheral nervous systems; asthma; glomerulosclerosis;septic shock and infection; and psoriasis.

In accordance with yet a further aspect of the present invention, thereis provided an antibody against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to human TIMP-4 sequences.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides which may be employed fortherapeutic purposes, for example, for remodeling and repairing tissueand for destruction of scar tissue.

In accordance with another aspect of the present invention, there areprovided diagnostic assays for detecting diseases related to mutationsin human TIMP-4 sequences and over-expression of the polypeptide.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1A-1B shows the cDNA sequence and corresponding deduced amino acidsequence of the full-length human TIMP-4 polypeptide. The standardone-letter abbreviations for amino acids are used. Sequencing wasperformed using a 373 Automated DNA sequencer (Applied Biosystems,Inc.). Sequencing accuracy is predicted to be greater than 97% accurate.

FIG. 2 is an amino acid sequence comparison between the polypeptide ofthe present invention and other human TIMP polypeptides.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 or for themature polypeptide encoded by the cDNA of the clone deposited as ATCCDeposit No. 75946 on Nov. 11, 1994. The ATCC (American Type CultureCollection) is located at 10801 University Boulevard, Manassas, Va.20110-2209.

A polynucleotide encoding a polypeptide encodes a protein of 224 aminoacid residues of which approximately the first 29 residues represent theleader sequence such that the mature protein comprises 195 amino acidresidues. The polynucleotide was discovered in an early stage humanbrain cDNA library. The protein exhibits the highest degree of homologyto Human TIMP-2 with 48% identity and 72% similarity over a 136 aminoacid stretch. Human TIMP-4 has the signature 12 cysteine amino acids.The 12 cysteine residues are involved in forming disulfide bonds whichare critical for folding into proper tertiary structure. Further, theputative active site is the first 22 amino acid residues past the signalpeptide which contains HIS_(7 and GLN)9.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIGS. 1A-1B or that of the deposited cloneor may be a different coding sequence which coding sequence, as a resultof the redundancy or degeneracy of the genetic code, encodes the same,mature polypeptide as the DNA of FIGS. 1A-1B or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIGS.1A-1B or for the mature polypeptide encoded by the deposited cDNA mayinclude: only the coding sequence for the mature polypeptide; the codingsequence for the mature polypeptide and additional coding sequence suchas a leader or secretory sequence or a proprotein sequence; the codingsequence for the mature polypeptide (and optionally additional codingsequence) and non-coding sequence, such as introns or non-codingsequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIGS. 1A-1B or the polypeptide encoded by the cDNA of the depositedclone. The variant of the polynucleotide may be a naturally occurringallelic variant of the polynucleotide or a non-naturally occurringvariant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIGS. 1A-1B or the same maturepolypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIGS. 1A-1B or thepolypeptide encoded by the cDNA of the deposited clone. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIGS. 1A-1B or of the coding sequence of the deposited clone.As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIGS. 1A-1B (SEQ ID NO:1)or the deposited cDNA(s).

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to a human TIMP-4 polypeptidewhich has the deduced amino acid sequence of FIGS. 1A-1B or which hasthe amino acid sequence encoded by the deposited cDNA, as well asfragments, analogs and derivatives of such polypeptide.

The terms “fragment”, “derivative” and “analog” when referring to thepolypeptide of FIGS. 1A-1B or that encoded by the deposited cDNA, meansa polypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIGS. 1A-1B orthat encoded by the deposited cDNA may be (i) one in which one or moreof the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the human TIMP-4 genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. Coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 and Sf9;animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plantcells, etc. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNHG16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacz, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonella tyhimuriumand various species within the genera Pseudomonas, Streptomyces, andStaphylococcus, although others may also be employed as a matter ofchoice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The human TIMP-4 polypeptides can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The present invention is also directed, in part, to human TIMP-4 whichhas, as a defining characteristic, the ability to inhibit the action ofMMP's. The human TIMP-4 polypeptide may be employed as ametalloproteinase inhibitor to prevent tumor invasion and angiogenesesand subsequent metastases. The human TIMP-4 polypeptide may also beemployed to treat arthritic diseases, such as rheumatoid arthritis andosteoarthritis, soft tissue rheumatism, polychondritis and tendonitis;and bone resorption diseases, such as osteoporosis, Paget's disease,hyperparathyroidism and cholesteatoma. Human TIMP-4 may also be employedto prevent enhanced collagen destruction that occurs in association withdiabetes, the recessive classes of dystrophic epidermolysis bullosa,periodontal disease and related consequences of gingival production ofcollagenase. human TIMP-4 may also be employed to inhibit PMNLcollagenase release following cellular infiltration to inflamed gingiva,including combatting the greater susceptibility of diabetes patients toperiodontal disease.

Human TIMP-4 may also be employed to treat corneal ulceration, forexample, that induced by alkali or other burns, by radiation, by VitaminE or retinoid deficiency; ulceration of the skin and gastro-intestinaltract, and abnormal wound healing, and post-operative conditionsincluding colonic anastomosis, in which collagenase levels are raised.

MMP's mediate tumor growth in situ. Accordingly, human TIMP-4 may beused to block the destruction of cellular basement membranes, which isthe mechanism by which cancer cells metastasize. MMP's have beenimplicated in neovascularization required to support tumor growth andsurvival, in the tissue remodeling required to accommodate the growingprimary and secondary tumors, and in the penetration of tumor cellsthrough the basement membranes of the vascular walls during metastasis.

MMP's are responsible for localized degradation of the follicular wallduring ovulation and localized degradation of the, uterine wall forblastocyte implantation. Accordingly, human TIMP-4 may be employed as acontraceptive.

Human TIMP-4 may also be employed as a general growth factor to treatrestenosis and similar diseases. Human TIMP-4 may be employedparticularly as a growth factor for erythroid cell lineages.

Among the other diseases which human TIMP-4 may be employed to treatincludes alveolitis, asthma, psoriasis, glomerulosclerosis, and septicshock since MMP's are involved in the tissue invasiveness of someparasites.

Fragments of the full length human TIMP-4 gene may be used as ahybridization probe for a cDNA library to isolate the full length geneand to isolate other genes which have a high sequence similarity to thegene or similar biological activity. Probes of this type can be, forexample, between 20 and 2000 bases. Preferably, however, the probes havebetween 30 and 50 base pairs. The probe may also be used to identify acDNA clone corresponding to a full length transcript and a genomic cloneor clones that contain the complete human TIMP-4 gene includingregulatory and promotor regions, exons, and introns. An example of ascreen comprises isolating the coding region of the human TIMP-4 gene byusing the known DNA sequence to synthesize an oligonucleotide probe.Labeled oligonucleotides having a sequence complementary to that of thegene of the present invention are used to screen a library of humancDNA, genomic DNA or mRNA to determine which members of the library theprobe hybridizes to.

This invention is also related to the use of the human TIMP-4 gene aspart of a diagnostic assay for detecting diseases or susceptibility todiseases related to the presence of mutated human TIMP-4.

Individuals carrying mutations in the human TIMP-4 gene may be detectedat the DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, such as from blood, urine,saliva, tissue biopsy and autopsy material. The genomic DNA may be useddirectly for detection or may be amplified enzymatically by using PCR(Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA orcDNA may also be used for the same purpose. As an example, PCR primerscomplementary to the nucleic acid encoding human TIMP-4 can be used toidentify and analyze human TIMP-4 mutations. For example, deletions andtn insertions can be detected by a change in size of the amplifiedproduct in comparison to the normal genotype. Point mutations can beidentified by hybridizing amplified DNA to radiolabeled human TIMP-4 RNAor alternatively, radiolabeled human TIMP-4 antisense DNA sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RPLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of human TIMP-4 protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease regulated by human TIMP-4. Assays used to detect levels of humanTIMP-4 protein in a sample derived from a host are well-known to thoseof skill in the art and include radioimmunoassays, competitive-bindingassays, Western Blot analysis, ELISA assays and “sandwich” assay. AnELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2),Chapter 6, (1991)) initially comprises preparing an antibody specific tothe human TIMP-4 antigen, preferably a monoclonal antibody. In additiona reporter antibody is prepared against the monoclonal antibody. To thereporter antibody is attached a detectable reagent such asradioactivity, fluorescence or, in this example, a horseradishperoxidase enzyme. A sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein like BSA. Next, the monoclonalantibody is incubated in the dish during which time the monoclonalantibodies attach to any human TIMP-4 proteins attached to thepolystyrene dish. All unbound monoclonal antibody is washed out withbuffer. The reporter antibody linked to horseradish peroxidase is nowplaced in the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to human TIMP-4. Unattached reporter antibodyis then washed out. Peroxidase substrates are then added to the dish andthe amount of color developed in a given time period is a measurement ofthe amount of human TIMP-4 protein present in a given volume of patientsample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to humanTIMP-4 are attached to a solid support and labeled human TIMP-4 and asample derived from the host are passed over the solid support and theamount of label detected, for example by liquid scintillationchromatography, can be correlated to a quantity of human TIMP-4 in thesample.

A “sandwich” assay is similar to an ELISA assay. In a “sandwich” assayhuman TIMP-4 is passed over a solid support and binds to antibodyattached to a solid support. A second antibody is then bound to thehuman TIMP-4. A third antibody which is labeled and specific to thesecond antibody is then passed over the solid support and binds to thesecond antibody and an amount can then be quantitated.

This invention also provides a method of screening compounds to identifythose which are agonists or antagonists to be human TIMP-4 polypeptide.An example of such a method comprises obtaining mammalian tissuecomprising an extra-cellular matrix, for example, bovine radiocarpaljoints. The articular cartilage is cut into smaller disks and labeledwith ³⁵S-sodium sulfate (10 micro Ci/ml) in DMEM for a sufficient timefor the cartilage to incorporate the labeled Sodium sulfate. An MMP, forexample, stromelysin, or IL1 or TNF is then added to the cartilage disksunder appropriate conditions such that tissue breakdown would normallyoccur. Human TIMP-4 and the compounds to be screened are then added tothe reaction mixture for a sufficient time for the MMP to normally breakdown the cartilage disks. The supernatant, which is the media outsidethe cartilage disks, is then collected and radioactivity is counted by aliquid scintillation counter. The percentage of ³⁵S released into themedia is then calculated. This release of ³⁵S-GAG is representative ofthe proteoglycan pool in the extracellular matrix of cartilage, andreflects proteoglycan degradation by the MMP. The amount of ³⁵S-GAG, asdetermined by liquid scintillation chromatography, is then compared to acontrol assay done in the absence of the compound to be screened and theability of the compound to agonize or antagonize the action of humanTIMP-4 may then be determined.

Examples of potential human TIMP-4 antagonists, in addition to thoseidentified above, include an antibody, or in some cases, anoligonucleotide, which binds to the polypeptide. Alternatively, apotential antagonist may be a mutated form of human TIMP-4, whichrecognizes natural substrates, but is inactive, and thereby prevent theaction of human TIMP-4.

Potential human TIMP-4 antagonists also include antisense constructsprepared using antisense technology. Antisense technology can be used tocontrol gene expression through triple-helix formation or antisense DNAor RNA, both of which methods are based on binding of a polynucleotideto DNA or RNA. For example, the 5′ coding portion of the polynucleotidesequence, which encodes for the mature polypeptides of the presentinvention, is used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcription(triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooneyet al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360(1991)), thereby preventing transcription and the production of humanTIMP-4. The antisense RNA oligonucleotide hybridizes to the mRNA in vivoand blocks translation of the mRNA molecule into the human TIMP-4(antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of human TIMP-4.

Another potential human TIMP-4 antagonist is a small molecule whichbinds to and occupies the active site of the human TIMP-4 therebypreventing human TIMP-4 from interacting with MMP's such that normalbiological activity is prevented. Examples of small molecules includebut are not limited to small peptides or peptide-like molecules, forexample a peptide-bonded molecule.

The human TIMP-4 antagonists may be employed for tissue repair andremodeling, for example, where destruction of scar tissue is desired. Insome situations, enhanced connective tissue turnover or remodeling maybe desirable, e.g. in resorption of scar tissue; in uterine involutionpost-partum; in remodeling of fibrotic deposits in the lung, liver orjoints. To appropriately control turnover of extra-cellular matrixproteins in these situations would require a balance between the MMP'sand human TIMP-4 to appropriately control degradation.

The polypeptides and agonists or antagonists that are also polypeptidesmay be employed in accordance with the present invention by expressionof such polypeptides in vivo, which is often referred to as “genetherapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, cells may be engineeredby procedures known in the art by use of a retroviral particlecontaining RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include; but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechnicues, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CKV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequencers) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells. Thepolypeptides and agonists or antagonists of the present invention may beemployed in combination with a suitable pharmaceutical carrier. Suchcompositions comprise a therapeutically effective amount of thepolypeptide, and a pharmaceutically acceptable carrier or excipient.Such a carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions may be employed in conjunction with othertherapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the topical, intravenous, intra-articular,intra-tumor, intraperitoneal, intramuscular, subcutaneous, intranasal orintradermal routes. The pharmaceutical compositions are administered inan amount which is effective for treating and/or prophylaxis of thespecific indication. In general, the pharmaceutical compositions areadministered in an amount of at least about 10 μg/kg body weight and inmost cases they will be administered in an amount not in excess of about8 mg/Kg body weight per day and preferably the dosage is from about 10μg/kg to about 1 mg/kg body weight daily, taking into account the routesof administration, symptoms, etc.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clones to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were. usedas would be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Bacterial Expression and Purification of human TIMP-4

The DNA sequence encoding for human TIMP-4, ATCC #75946, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ andsequences of the processed human TIMP-4 protein (minus the signalpeptide sequence) and the vector sequences 3′ to the TIMP-4 gene.Additional nucleotides corresponding to human TIMP-4 were added to the5′ and 3′ sequences respectively. The 5′ oligonucleotide primer has thesequence 5′ GCCAGAGGATCCTGCAGCTGCGCCCCGGCGCAC 3′ contains a BamH1restriction enzyme site followed by 21 nucleotides of human TIMP-4coding sequence starting from the presumed terminal amino acid of theprocessed protein codon. The 3′ sequence 5′CGGCTTCTAGAACTAGGGCTGAACGATGTCAAC 3′ contains an XbaI site and isfollowed by 18 nucleotides of human TIMP-4. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311).pQE-9 encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 was then digested with BamH1 and XbaI. The amplified sequenceswere ligated into pQE-9 and were inserted in frame with the sequenceencoding for the histidine tag and the RBS. The ligation mixture wasthen used to transform E. coli strain m15/pREP4 available from Qiagen bythe procedure described in Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989). m15/pREP4contains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(r)) .Transformants are identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies were selected. Plasmid DNA wasisolated and confirmed by restriction analysis. Clones containing thedesired constructs were grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells were grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) wasthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells were grown an extra 3 to 4 hours. Cells were thenharvested by centrifugation. The cell pellet was solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedhuman TIMP-4 was purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984). Human TIMP-4 (90% pure) was eluted from the columnin 6 molar guanidine HCl pH 5.0 and for the purpose of renaturationadjusted to 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mmolarglutathione (reduced) and 2 mmolar glutathione (oxidized). Afterincubation in this solution for 12 hours the protein was dialyzed to 10mmolar sodium phosphate.

EXAMPLE 2 Expression of Recombinant human TIMP-4 in COS Cells

The expression of human TIMP-4 HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the entire human TIMP-4 precursor and a HA tag fusedin frame to its 3′ end was cloned into the polylinker region of thevector, therefore, the recombinant protein expression is directed underthe CMV promoter. The HA tag correspond to an epitope derived from theinfluenza hemagglutinin protein as previously described (I. Wilson, H.Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell37, 767). The fusion of HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence ATCC #75946, encoding for human TIMP-4 was constructedby PCR using two primers: the 5′ primer 5′GCCAGAGGATCCGCCACCATGCCTGGGAGCCCTCGGCCC 3′ contains a BaMHI sitefollowed by 21 nucleotides of human TIMP-4 coding sequence starting fromthe initiation codon; the 3′ sequence 5′CGGCTCTAGAATCAAGCGTAGTCTGGGACGTCGTATGGGTAGGGCTGAACGATG TCAAC 3′ containscomplementary sequences to an XbaI site, translation stop codon, HA tagand the last 18 nucleotides of the human TIMP-4 coding sequence (notincluding the stop codon). Therefore, the PCR product contains a BamHIsite, human TIMP-4 coding sequence followed by HA tag fused in frame, atranslation termination stop codon next to the HA tag, and an XbaI site.The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digestedwith BamHI and XbaI restriction enzyme and ligated. The ligation mixturewas transformed into E. coli strain SURE (available from StratageneCloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037)the transformed culture was plated on ampicillin media plates andresistant colonies were selected. Plasmid DNA was isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant human TIMP-4,COS cells were transfected with the expression vector by DEAB-DEXTRANmethod (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989)). The expressionof the human TIMP-4 HA protein was detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells werelabelled for 8 hours with ³⁵S-cysteine two days post transfection.Culture media were then collected and cells were lysed with detergent(RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mMTris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysateand culture media were precipitated with a HA specific monoclonalantibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.

EXAMPLE 3 Cloning and Expression of TIMP-4 Using the BaculovirusExpression System

The DNA sequence encoding the full length TIMP-4 protein, ATCC #75946,was amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene:

The 5′ primer has the sequence 5′ GCCAGAGGATCCATGCCTGG GAGCCCTCGGCCC 3′and contains a BamHI restriction enzyme site (in bold) just behind thefirst 21 nucleotides of the TIMP-4 gene (the initiation codon fortranslation “ATG” is underlined).

The 3′ primer has the sequence 5′ CGGCTTCTAGAACTAGGGCTG AACGATGTCAAC 3′and contains the cleavage site for the restriction endonuclease XbaI and18 nucleotides complementary to the 3′ non-translated sequence of theTIMP-4 gene. The amplified sequences were isolated from a 1% agarose gelusing a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment was then digested with the endonucleases BamHI andXbaI and then purified again on a 1% agarose gel. This fragment isdesignated F2.

The vector pA2 (modification of pVL941 vector, discussed below) is usedfor the expression of the TIMP-4 protein using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).This expression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhidrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI and XbaI.The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid was digested with the restriction enzymes BamHI and XbaI.The DNA was then isolated from a 1% agarose gel using the commerciallyavailable kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vectorDNA is designated V2.

Fragment F2 and the plasmid V2 were ligated with T4 DNA ligase. E. coliHB101 cells were then transformed and bacteria identified that containedthe plasmid (pBacTIMP-4) with the TIMP-4 gene using the enzymes BamHIand XbaI. The sequence of the cloned fragment was confirmed by DNAsequencing.

5 μg of the plasmid pBacTIMP-4 was co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacTIMP-4 weremixed in a sterile well of a microtiter plate containing 50 μl of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27° C. After 5hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at27° C. for four days.

After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses were added to the cellsand blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculovirus was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

Sf9 tells were grown in Grace's medium supplemented with 10%heat-inactivated PBS. The cells were infected with the recombinantbaculovirus V-TIMP-4 at a multiplicity of infection (MOI) of 2. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 4 Expression Pattern of Human TIMP-4 in Human Tissues

20 μg of total RNA from each of the above tissues was denatured and runon a 1.2% formaldehyde agarose gel and capillary blotted onto a nylonfilter overnight. RNA was immobilized on the filter by UV cross-linking.A random primer probe was prepared from the EcoRI-Xhol insert of thepartial TIMP-4 nucleic acid sequence and used to probe the blot byovernight hybridization in Church buffer with 100 μg/ml denaturedherring sperm DNA as a blocking agent. Washing was performedsequentially with 2×SSC/0.1% SDA and 0.2×SSC/0.1% SDS at 65 degreesCelsius. Size markers were the BRL RNA ladder and 18S and 28S ribosomalRNA bands.

EXAMPLE 5 Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time; fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer containing an EcoRI site and the 3′ primerfurther includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS) penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

What is claimed is:
 1. A method for the treatment of a patient havingneed of TIMP-4 comprising: administering to the patient atherapeutically effective amount of a polypeptide selected from thegroup consisting of: (a) a polypeptide having the deduced amino acidsequence of SEQ ID NO:2 and fragments thereof; (b) a polypeptidecomprising amino acid 1 to amino acid 195 of SEQ ID NO:2; and (c) apolypeptide encoded by the cDNA of ATCC Deposit No. 75946 and fragmentsof said polypeptide; wherein said polypeptide and said fragments inhibitmetalloproteinase activity.
 2. A method of inhibiting metalloproteinaseactivity which comprises, contacting a metalloproteinase with an amountof a polypeptide effective to inhibit metalloproteinase activity,wherein said polypeptide has an amino acid sequence selected from thegroup consisting of: (a) at least 30 amino acids of the amino acidsequence shown as residues 1 to 224 of SEQ ID NO:2, wherein said atleast 30 amino acids retain metalloproteinase inhibiting activity; (b) afragment of the amino acid sequence shown as residues 1 to 224 of SEQ IDNO:2, wherein said fragment retains metalloproteinase inhibitingactivity; (c) the amino acid sequence of a polypeptide which possessesmetalloproteinase inhibiting activity wherein said amino acid sequenceis encoded by a polynucleotide which hybridizes to a DNA consisting of asequence complementary to SEQ ID NO:1 when incubated in Church bufferwith 100 ug/ml denatured herring sperm DNA as a blocking agent followedby washing sequentially with 2×SSC/0.1% SDS and 0.2×SSC/0.1% SDS at 65°C.; (d) a first amino acid sequence at least 90% identical to a secondamino acid sequence shown as residues 30 to 224 in SEQ ID NO:2, whereinsaid first amino acid sequence retains metalloproteinase inhibitingactivity; (e) at least 30 amino acids of the amino acid sequence of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said at least 30 amino acids retainmetalloproteinase inhibiting activity; and (f) a fragment of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said fragment retains metalloproteinaseinhibiting activity.
 3. The method of claim 2, wherein said polypeptidecomprises amino acid sequence (a).
 4. The method of claim 3, whereinsaid polypeptide comprises the amino acid sequence shown as residues 30to 224 in SEQ ID NO:2.
 5. The method of claim 3, wherein saidpolypeptide comprises the amino acid sequence shown as residues 2 to 224in SEQ ID NO:2.
 6. The method of claim 3, wherein said polypeptidecomprises the amino acid sequence shown as residues 1 to 224 in SEQ IDNO:2.
 7. The method of claim 2, wherein said polypeptide comprises aminoacid sequence (b).
 8. The method of claim 2, wherein said polypeptidecomprises amino acid sequence (c).
 9. The method of claim 2, whereinsaid polypeptide comprises amino acid sequence (d).
 10. The method ofclaim 2, wherein said polypeptide comprises amino acid sequence (e). 11.The method of claim 10, wherein said polypeptide comprises the aminoacid sequence of the mature polypeptide encoded by the human cDNAcontained in ATCC Deposit No.
 75946. 12. The method of claim 10, whereinsaid polypeptide comprises the amino acid sequence of the full-lengthpolypeptide encoded by the human cDNA contained in ATCC Deposit No.75946, excepting the N-terminal methionine.
 13. The method of claim 10,wherein said polypeptide comprises the amino acid sequence of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No.
 75946. 14. The method of claim 2, wherein said polypeptidecomprises amino acid sequence (f).
 15. The method of claim 2, whereinsaid polypeptide comprises a heterologous polypeptide sequence.
 16. Themethod of claim 2, wherein said polypeptide is in a pharmaceuticallyacceptable carrier.
 17. A method of inhibiting the destruction ofcellular basement membranes in a patient which comprises, administeringto the patient, a therapeutically effective amount of a polypeptidehaving an amino acid sequence selected from the group consisting of: (a)at least 30 amino acids of the amino acid sequence shown as residues 1to 224 of SEQ ID NO:2, wherein said at least 30 amino acids retainmetalloproteinase inhibiting activity; (b) a fragment of the amino acidsequence shown as residues 1 to 224 of SEQ ID NO:2, wherein saidfragment retains metalloproteinase inhibiting activity; (c) the aminoacid sequence of a polypeptide which possesses metalloproteinaseinhibiting activity wherein said amino acid sequence is encoded by apolynucleotide which hybridizes to a DNA consisting of a sequencecomplementary to SEQ ID NO:1 when incubated in Church buffer with 100ug/ml denatured herring sperm DNA as a blocking agent followed bywashing sequentially with 2×SSC/0.1% SDS and 0.2×SSC/0.1% SDS at 65° C.;(d) a first amino acid sequence at least 90% identical to a second aminoacid sequence shown as residues 30 to 224 in SEQ ID NO:2, wherein saidfirst amino acid sequence retains metalloproteinase inhibiting activity;(e) at least 30 amino acids of the amino acid sequence of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said at least 30 amino acids retainmetalloproteinase inhibiting activity; and (f) a fragment of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said fragment retains metalloproteinaseinhibiting activity.
 18. The method of claim 17, wherein saidpolypeptide comprises amino acid sequence (a).
 19. The method of claim18, wherein said polypeptide comprises the amino acid sequence shown asresidues 30 to 224 in SEQ ID NO:2.
 20. The method of claim 18, whereinsaid polypeptide comprises the amino acid sequence shown as residues 2to 224 in SEQ ID NO:2.
 21. The method of claim 18, wherein saidpolypeptide comprises the amino acid sequence shown as residues 1 to 224in SEQ ID NO:2.
 22. The method of claim 17, wherein said polypeptidecomprises amino acid sequence (b).
 23. The method of claim 17, whereinsaid polypeptide comprises amino acid sequence (c).
 24. The method ofclaim 17, wherein said polypeptide comprises amino acid sequence (d).25. The method of claim 17, wherein said polypeptide comprises aminoacid sequence (e).
 26. The method of claim 25, wherein said polypeptidecomprises the amino acid sequence of the mature polypeptide encoded bythe human cDNA contained in ATCC Deposit No.
 75946. 27. The method ofclaim 25, wherein said polypeptide comprises the amino acid sequence ofthe full-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, excepting the N-terminal methionine.
 28. The methodof claim 25, wherein said polypeptide comprises the amino acid sequenceof the full-length polypeptide encoded by the human cDNA contained inATCC Deposit No.
 75946. 29. The method of claim 17, wherein saidpolypeptide comprises amino acid sequence (f).
 30. The method of claim17, wherein said polypeptide comprises a heterologous polypeptidesequence.
 31. The method of claim 17, wherein said polypeptide is in apharmaceutically acceptable carrier.
 32. A method of treating restenosiswhich comprises, administering to a patient, a therapeutically effectiveamount of polypeptide having an amino acid sequence selected from thegroup consisting of: (a) at least 30 amino acids of the amino acidsequence shown as residues 1 to 224 of SEQ ID NO:2, wherein said atleast 30 amino acids retain metalloproteinase inhibiting activity; (b) afragment of the amino acid sequence shown as residues 1 to 224 of SEQ IDNO:2, wherein said fragment retains metalloproteinase inhibitingactivity; (c) the amino acid sequence of polypeptide which possessesmetalloproteinase inhibiting activity wherein said amino acid sequenceis encoded by a polynucleotide which hybridizes to a DNA consisting of asequence complementary to SEQ ID NO:1 when incubated in Church bufferwith 100 ug/ml denatured herring sperm DNA as a blocking agent followedby washing sequentially with 2×SSC/0.1% SDS and 0.2×SSC/0.1% SDS at 65°C.; (d) a first amino acid sequence at least 90% identical to a secondamino acid sequence shown as residues 30 to 224 in SEQ ID NO:2, whereinsaid first amino acid sequence retains metalloproteinase inhibitingactivity; (e) at least 30 amino acids of the amino acid sequence of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said at least 30 amino acids retainmetalloproteinase inhibiting activity; and (f) a fragment of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said fragment retains metalloproteinaseinhibiting activity.
 33. The method of claim 32, wherein saidpolypeptide comprises amino acid sequence (a).
 34. The method of claim33, wherein said polypeptide comprises the amino acid sequence shown asresidues 30 to 224 in SEQ ID NO:2.
 35. The method of claim 33, whereinsaid polypeptide comprises the amino acid sequence shown as residues 2to 224 in SEQ ID NO:2.
 36. The method of claim 33, wherein saidpolypeptide comprises the amino acid sequence shown as residues 1 to 224in SEQ ID NO:2.
 37. The method of claim 32, wherein said polypeptidecomprises amino acid sequence (b).
 38. The method of claim 32, whereinsaid polypeptide comprises amino acid sequence (c).
 39. The method ofclaim 32, wherein said polypeptide comprises amino acid sequence (d).40. The method of claim 32, wherein said polypeptide comprises aminoacid sequence (e).
 41. The method of claim 40, wherein said polypeptidecomprises the amino acid sequence of the mature polypeptide encoded bythe human cDNA contained in ATCC Deposit No.
 75946. 42. The method ofclaim 40, wherein said polypeptide comprises the amino acid sequence ofthe full-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, excepting the N-terminal methionine.
 43. The methodof claim 40, wherein said polypeptide comprises the amino acid sequenceof the full-length polypeptide encoded by the human cDNA contained inATCC Deposit No.
 75946. 44. The method of claim 32, wherein saidpolypeptide comprises amino acid sequence (f).
 45. The method of claim32, wherein said polypeptide comprises a heterologous polypeptidesequence.
 46. The method of claim 32, wherein said polypeptide is in apharmaceutically acceptable carrier.
 47. A method of treating cancerwhich comprises, administering to a patient, a therapeutically effectiveamount of a polypeptide having an amino acid sequence selected from thegroup consisting of: (a) at least 30 amino acids of the amino acidsequence shown as residues 1 to 224 of SEQ ID NO:2, wherein said atleast 30 amino acids retain metalloproteinase inhibiting activity; (b) afragment of the amino acid sequence shown as residues 1 to 224 of SEQ IDNO:2, wherein said fragment retains metalloproteinase inhibitingactivity; (c) the amino acid sequence of a polypeptide which possessesmetalloproteinase inhibiting activity wherein said amino acid sequenceis encoded by a polynucleotide which hybridizes to a DNA consisting of asequence complementary to SEQ ID NO:1 when incubated in Church bufferwith 100 ug/ml denatured herring sperm DNA as a blocking agent followedby washing sequentially with 2×SSC/0.1% SDS and 0.2×SSC/0.1% SDS at 65°C.; (d) a first amino acid sequence at least 90% identical to a secondamino acid sequence shown as residues 30 to 224 in SEQ ID NO:2, whereinsaid first amino acid sequence retains metalloproteinase inhibitingactivity; (e) at least 30 amino acids of the amino acid sequence of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said at least 30 amino acids retainmetalloproteinase inhibiting activity; and (f) a fragment of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said fragment retains metalloproteinaseinhibiting activity.
 48. The method of claim 47, wherein saidpolypeptide comprises amino acid sequence (a).
 49. The method of claim48, wherein said polypeptide comprises the amino acid sequence shown asresidues 30 to 224 in SEQ ID NO:2.
 50. The method of claim 48, whereinsaid polypeptide comprises the amino acid sequence shown as residues 2to 224 in SEQ ID NO:2.
 51. The method of claim 48, wherein saidpolypeptide comprises the amino acid sequence shown as residues 1 to 224in SEQ ID NO:2.
 52. The method of claim 47, wherein said polypeptidecomprises amino acid sequence (b).
 53. The method of claim 47, whereinsaid polypeptide comprises amino acid sequence (c).
 54. The method ofclaim 47, wherein said polypeptide comprises amino acid sequence (d).55. The method of claim 47, wherein said polypeptide comprises aminoacid sequence (e).
 56. The method of claim 55, wherein said polypeptidecomprises the amino acid sequence of the mature polypeptide encoded bythe human cDNA contained in ATCC Deposit No.
 75946. 57. The method ofclaim 55, wherein said polypeptide comprises the amino acid sequence ofthe full-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, excepting the N-terminal methionine.
 58. The methodof claim 55, wherein said polypeptide comprises the amino acid sequenceof the full-length polypeptide encoded by the human cDNA contained inATCC Deposit No.
 75946. 59. The method of claim 47, wherein saidpolypeptide comprises amino acid sequence (f).
 60. The method of claim47, wherein said polypeptide comprises a heterologous polypeptidesequence.
 61. The method of claim 47, wherein said polypeptide is in apharmaceutically acceptable carrier.
 62. A method of inhibiting tumorinvasion which comprises, administering to a patient, a therapeuticallyeffective amount of a polypeptide having an amino acid sequence selectedfrom the group consisting of: (a) at least 30 amino acids of the aminoacid sequence shown as residues 1 to 224 of SEQ ID NO:2, wherein said atleast 30 amino acids retain metalloproteinase inhibiting activity; (b) afragment of the amino acid sequence shown as residues 1 to 224 of SEQ IDNO:2, wherein said fragment retains metalloproteinase inhibitingactivity; (c) the amino acid sequence of a polypeptide which possessesmetalloproteinase inhibiting activity wherein said amino acid sequenceis encoded by a polynucleotide which hybridizes to a DNA consisting of asequence complementary to SEQ ID NO:1 when incubated in Church bufferwith 100 ug/ml denatured herring sperm DNA as a blocking agent followedby washing sequentially with 2×SSC/0.1% SDS and 0.2×SSC/0.1% SDS at 65°C.; (d) a first amino acid sequence at least 90% identical to a secondamino acid sequence shown as residues 30 to 224 in SEQ ID NO:2, whereinsaid first amino acid sequence retains metalloproteinase inhibitingactivity; (e) at least 30 amino acids of the amino acid sequence of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said at least 30 amino acids retainmetalloproteinase inhibiting activity; and (f) a fragment of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said fragment retains metalloproteinaseinhibiting activity.
 63. The method of claim 62, wherein saidpolypeptide comprises amino acid sequence (a).
 64. The method of claim63, wherein said polypeptide comprises the amino acid sequence shown asresidues 30 to 224 in SEQ ID NO:2.
 65. The method of claim 63, whereinsaid polypeptide comprises the amino acid sequence shown as residues 2to 224 in SEQ ID NO:2.
 66. The method of claim 63, wherein saidpolypeptide comprises the amino acid sequence shown as residues 1 to 224in SEQ ID NO:2.
 67. The method of claim 62, wherein said polypeptidecomprises amino acid sequence (b).
 68. The method of claim 62, whereinsaid polypeptide comprises amino acid sequence (c).
 69. The method ofclaim 62, wherein said polypeptide comprises amino acid sequence (d).70. The method of claim 62, wherein said polypeptide comprises aminoacid sequence (e).
 71. The method of claim 70, wherein said polypeptidecomprises the amino acid sequence of the mature polypeptide encoded bythe human cDNA contained in ATCC Deposit No.
 75946. 72. The method ofclaim 70, wherein said polypeptide comprises the amino acid sequence ofthe full-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, excepting the N-terminal methionine.
 73. The methodof claim 70, wherein said polypeptide comprises the amino acid sequenceof the full-length polypeptide encoded by the human cDNA contained inATCC Deposit No.
 75946. 74. The method of claim 62, wherein saidpolypeptide comprises amino acid sequence (f).
 75. The method of claim62, wherein said polypeptide comprises a heterologous polypeptidesequence.
 76. The method of claim 62, wherein said polypeptide is in apharmaceutically acceptable carrier.
 77. A method of inhibitingangiogenesis which comprises, administering to a patient, atherapeutically effective amount of a polypeptide having an amino acidsequence selected from the group consisting of: (a) at least 30 aminoacids of the amino acid sequence shown as residues 1 to 224 of SEQ IDNO:2, wherein said at least 30 amino acids retain metalloproteinaseinhibiting activity; (b) a fragment of the amino acid sequence shown asresidues 1 to 224 of SEQ ID NO:2, wherein said fragment retainsmetalloproteinase inhibiting activity; (c) the amino acid sequence of apolypeptide which possesses metalloproteinase inhibiting activitywherein said amino acid sequence is encoded by a polynucleotide whichhybridizes to a DNA consisting of a sequence complementary to SEQ IDNO:1 when incubated in Church buffer with 100 ug/ml denatured herringsperm DNA as a blocking agent followed by washing sequentially with2×SSC/0.1% SDS and 0.2×SSC/0.1% SDS at 65° C.; (d) a first amino acidsequence at least 90% identical to a second amino acid sequence shown asresidues 30 to 224 in SEQ ID NO:2, wherein said first amino acidsequence retains metalloproteinase inhibiting activity; (e) at least 30amino acids of the amino acid sequence of the full-length polypeptideencoded by the human cDNA contained in ATCC Deposit No. 75946, whereinsaid at least 30 amino acids retain metalloproteinase inhibitingactivity; and (f) a fragment of the full-length polypeptide encoded bythe human cDNA contained in ATCC Deposit No. 75946, wherein saidfragment retains metalloproteinase inhibiting activity.
 78. The methodof claim 77, wherein said polypeptide comprises amino acid sequence (a).79. The method of claim 78, wherein said polypeptide comprises the aminoacid sequence shown as residues 30 to 224 in SEQ ID NO:2.
 80. The methodof claim 78, wherein said polypeptide comprises the amino acid sequenceshown as residues 2 to 224 in SEQ ID NO:2.
 81. The method of claim 78,wherein said polypeptide comprises the amino acid sequence shown asresidues 1 to 224 in SEQ ID NO:2.
 82. The method of claim 77, whereinsaid polypeptide comprises amino acid sequence (b).
 83. The method ofclaim 77, wherein said polypeptide comprises amino acid sequence (c).84. The method of claim 77, wherein said polypeptide comprises aminoacid sequence (d).
 85. The method of claim 77, wherein said polypeptidecomprises amino acid sequence (e).
 86. The method of claim 85, whereinsaid polypeptide comprises the amino acid sequence of the maturepolypeptide encoded by the human cDNA contained in ATCC Deposit No.75946.
 87. The method of claim 85, wherein said polypeptide comprisesthe amino acid sequence of the full-length polypeptide encoded by thehuman cDNA contained in ATCC Deposit No. 75946, excepting the N-terminalmethionine.
 88. The method of claim 85, wherein said polypeptidecomprises the amino acid sequence of the full-length polypeptide encodedby the human cDNA contained in ATCC Deposit No.
 75946. 89. The method ofclaim 77, wherein said polypeptide comprises amino acid sequence (f).90. The method of claim 77, wherein said polypeptide comprises aheterologous polypeptide sequence.
 91. The method of claim 77, whereinsaid polypeptide is in a pharmaceutically acceptable carrier.
 92. Amethod of inhibiting metastasis which comprises, administering to apatient, a therapeutically effective amount of a polypeptide having anamino acid sequence selected from the group consisting of: (a) at least30 amino acids of the amino acid sequence shown as residues 1 to 224 ofSEQ ID NO:2, wherein said at least 30 amino acids retainmetalloproteinase inhibiting activity; (b) a fragment of the amino acidsequence shown as residues 1 to 224 of SEQ ID NO:2, wherein saidfragment retains metalloproteinase inhibiting activity; (c) the aminoacid sequence of a polypeptide which possesses metalloproteinaseinhibiting activity wherein said amino acid sequence is encoded by apolynucleotide which hybridizes to a DNA consisting of a sequencecomplementary to SEQ ID NO:1 when incubated in Church buffer with 100ug/ml denatured herring sperm DNA as a blocking agent followed bywashing sequentially with 2×SSC/0.1% SDS and 0.2×SSC/0.1% SDS at 65° C.;(d) a first amino acid sequence at least 90% identical to a second aminoacid sequence shown as residues 30 to 224 in SEQ ID NO:2, wherein saidfirst amino acid sequence retains metalloproteinase inhibiting activity;(e) at least 30 amino acids of the amino acid sequence of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said at least 30 amino acids retainmetalloproteinase inhibiting activity; and (f) a fragment of thefull-length polypeptide encoded by the human cDNA contained in ATCCDeposit No. 75946, wherein said fragment retains metalloproteinaseinhibiting activity.
 93. The method of claim 92, wherein saidpolypeptide comprises amino acid sequence (a).
 94. The method of claim93, wherein said polypeptide comprises the amino acid sequence shown asresidues 30 to 224 in SEQ ID NO:2.
 95. The method of claim 93, whereinsaid polypeptide comprises the amino acid sequence shown as residues 2to 224 in SEQ ID NO:2.
 96. The method of claim 93, wherein saidpolypeptide comprises the amino acid sequence shown as residues 1 to 224in SEQ ID NO:2.
 97. The method of claim 92, wherein said polypeptidecomprises amino acid sequence (b).
 98. The method of claim 92, whereinsaid polypeptide comprises amino acid sequence (c).
 99. The method ofclaim 92, wherein said polypeptide comprises amino acid sequence (d).100. The method of claim 92, wherein said polypeptide comprises aminoacid sequence (e).
 101. The method of claim 100, wherein saidpolypeptide comprises the amino acid sequence of the mature polypeptideencoded by the human cDNA contained in ATCC Deposit No.
 75946. 102. Themethod of claim 100, wherein said polypeptide comprises the amino acidsequence of the full-length polypeptide encoded by the human cDNAcontained in ATCC Deposit No. 75946, excepting the N-terminalmethionine.
 103. The method of claim 100, wherein said polypeptidecomprises the amino acid sequence of the full-length polypeptide encodedby the human cDNA contained in ATCC Deposit No.
 75946. 104. The methodof claim 92, wherein said polypeptide comprises amino acid sequence (f).105. The method of claim 92, wherein said polypeptide comprises aheterologous polypeptide sequence.
 106. The method of claim 92, whereinsaid polypeptide is in a pharmaceutically acceptable carrier.